Characterization and Antibacterial Activity of Phthalides from the Roots of the Medicinal Herb Levisticum officinale W.D.J. Koch.

A new phthalide, namely 7-methoxy-3-propylidenephthalide (1), along with two known compounds (2, 3) were isolated from the roots of the edible herb Levisticum officinale W.D.J. Koch, commonly known as lovage and well known in traditional medicine for its spasmolytic and diuretic effects. The structure of the new compound was established by HRMS and 1D & 2D NMR (1H 1H COSY, HMQC, and HMBC) spectroscopic analysis. All compounds are reported for the first time from L. officinale. Compounds 1-3 were tested against two Gram negative (Escherichia coli, Pseudomonas aeruginosa) and two Gram positive (Staphylococcus aureus and vancomycin-resistant Enterococcus [VRE] faecium) bacteria strains. Compound 3 was active against S. aureus, E. coli and vancomycin-resistant E. faecium with MIC values of 16, 64, and 128 μg/mL, respectively.


Introduction
Phthalides are a class of secondary metabolites with a wide range of pharmacological activities including inhibition of various enzymes, antiinflammatory, anti-atherosclerosis, blood viscosity reduction, anti-angina, anti-convulsion, and antimicrobial activities (1)(2)(3)(4). Phthalides and their derivatives including dihydro, tetrahydro, and dimeric compounds have been found in several species of the Apiaceae family (2).
L. officinale grows widely in the Hezar Mountain of the Kerman Province in Iran. The root of this plant has been used for various biological effects primarily as a diuretic, as a treatment for urinary tract infection and as a spasmolytic agent (6)(7)(8).
In order to discover new and potentially bioactive constituent from L. officinale, we investigated the ethyl acetate extract of the root. In this study, we report on the isolation, structural identification, and antibacterial activity of three phthalides, including a new one.

Experimental
General experimental procedures NMR spectra were recorded on a Bruker AVANCE III spectrometer operating at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR. UHPLC-MS analyses were performed on a Waters Acquity UPLC system coupled to a Waters Xevo TM quadrupole time-of-flight (QToF) mas spectrometer and equipped with an electrospray source with lockspray interface for accurate mass measurements. Silica gel (70-230 mesh) was used for column chromatography, and precoated silica gel F 254 (20 × 20 cm) plates for TLC, both supplied by the Merck. C 18 -reversed phase silica gel used for column chromatography, was purchased from Sigma.

Plant material
The roots of L. officinale were collected in July 2015 from the Hezar Mountain of Kerman Province, Iran. The plant material was identified by Prof. Farideh Attar from Tehran University. A voucher specimen (46553-TUH) has been deposited in the herbarium of the Science Faculty of Tehran University, Iran.

Extraction and isolation
Air-dried, powdered roots of L. officinale (3 kg) were extracted with n-hexane (3 x 9 L) and then ethyl acetate (3 x 9 L) by maceration at room temperature. The ethyl acetate extract was concentrated by rotary evaporation, to afford 100 g of dried extract. This extract was subjected to silica gel column chromatography (230-400 mesh, 1 kg), with a gradient of n-hexane-EtOAc and then EtOAc-MeOH as the eluent to give 16 fractions. Fraction 6 (1.8 g) was separated on a silica gel column (230-400 mesh) into five subfractions (6a-6e). Subfraction 6a (50 mg) was applied to a reverse phase silica gel column (8 g) and eluted with methanol-water as eluent, to afford compound 1 (2 mg). Purification of subfraction 6b (40 mg) on a reverse phase silica gel (7 g) column with a gradient of methanol-water as eluent led to the isolation of compound 2 (3 mg). Subfraction 6c (70 mg) was purified on a reverse phase silica (12 g) column to give compound 3 (3.5 mg).   adjusted to 0.5 McFarland standard turbidity and were further diluted (1:100) using MHB medium just before adding to the serially diluted samples. The plates were incubated for 24 h at 37 °C. MIC values were recorded as the lowest concentrations, which could inhibit the visible growth of the microorganisms. Each experiment was done in triplicate.

Result and Discussion
Phytochemical analysis of the ethyl acetate extract from L. officinale led to isolation and identification of a new phthalide (1), together with two knowns compounds namely, 5-hydroxybutylidene phthalide (2) and 7-hydroxybutylidene phthalide (3) (Figure 1).  (Table 1) were resolved into two methyl (corresponding to one methoxy and one aliphatic methyl), one methylene, four methine and five quaternary carbons. The 1 H NMR data (Table 1)  The NMR spectroscopic data of 1 showed high similarity to those of 3, previously isolated from Petroselinum crispum [9], indicating that they are structurally related. Comparison of the 1 H NMR and 13 C NMR data of these two compounds indicated the loss of a CH 2 group in side chain of compound 1, which was also evidenced by the 1 H-1 H COSY and HMBC spectral data of compound 1 (Figure 2). Furthermore, an additional methyl signal was observed at δ H /δ C 4.01/56.1, indicating the replacement of the phenolic hydroxyl group by a methoxy group. This was confirmed by a HMBC correlation from the methyl group to C-7. Thus, compound 1 was ascribed the name 7-methoxy-3-propylidenephthalide. The structure of the known compounds (2 and 3) was identified using 1 H-NMR and 13 C-NMR, as well as by comparison the data with those reported in literature (9).
Apiaceae plants are rich source for phthalide compounds such as dimer and simple phthalides. Among simple phthalides, butylphthalides are the most abundant, but propylphthalides are rather rare (2,3). So, presence of compound 1 as propylphthalide in L. officinale is notable.
The activity of the isolated compounds was studied against strains of the  (Table 2). Thus, it appears that the free hydroxyl group is required for antibacterial activity in these derivatives.  (3). For example, ligustilide has previously been reported for its growth inhibitory activity against E. coli (MIC = 4 mg/ mL), and S. aureus (1 mg/mL). Antibacterial activity was found for butylidenephthalide against E. coli and S. aureus with MIC values of 4 mg/mL, while (Z)-ligustilide exhibited moderate antibacterial activity against S. aureus with MIC value of 1 mg/mL (3).

Conclusion
In the present study, three phthalide derivatives were isolated from the roots of L. officinale, including new one. Their structures were elucidated by means of extensive 1D, 2D NMR spectroscopy. Also, the antibacterial activity of the compounds was studied against two Gram negative and two Gram positive bacteria strains. Among them, compound 3 hold a good potential for use in future studies due to their antibacterial properties. To our knowledge, this is the first report on the isolation and structure elucidation of the chemical constituents of L. officinale in Iran. All compounds are reported for the first time from L. officinale. Simple phthalides are not widely reported as showing any significant antibacterial activity. Therefore, the antibacterial activity of compound 3 is notable.
Supplementary material 1D and 2D NMR spectra of compound 1 can be found as Supporting Information.